Wholly Rickettsia! Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells

Item Type Poster/Presentation

Authors Driscoll, Timothy P.; Verhoeve, Victoria I.; Guillotte, Mark L.; Lehman, Stephanie S.; Rennoll, Sherri A.; Beier-Sexton, Magda; Rahman, M. Sayeedur; Azad, Abdu F.; Gillespie, Joseph J.

Publication Date 2018-06

Keywords Metabolic Networks and Pathways; Rickettsia--genetics; Rickettsia--metabolism

Download date 01/10/2021 05:45:11

Link to Item http://hdl.handle.net/10713/16003 Wholly Rickettsia! Metabolic Profile of the Quintessential Bacterial Parasite of Eukaryotic Cells Timothy Driscoll a a b b b b b b b Timothy P. Driscoll , Victoria I. Verhoeve , Mark L. Guillotte , Stephanie S. Lehman , Sherri A. Rennoll , Magda Beier-Sexton , M. Sayeedur Rahman , Abdu F. Azad , and Joseph J. Gillespie [email protected] Department of Biology, West Virginia University, Morgantown, WV 26505, USAa; Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USAb @driscollMML (twitter, fb, reddit, github)

BACKGROUND RESULTS Figure 2. Synopsis of known and predicted metabolites imported from Figure 3. Fatty acids and glycerophospholipids are synthesized Figure 5A. Cell envelope glycoconjugates are synthesized from Bacteria in the genus Rickettsia (Rickettsiaceae; Alphaproteobacteria) Our metabolic reconstruction (Fig. 1) identified 51 host metabolites (including 21 the eukaryotic cytoplasm by rickettsiae. from host precursors dephospho-CoA and , DHAP and G3P. imported host sugars D-ribose 5-P, UDP-glucose, and NAG-1-P. known to be imported Dephospho-CoA Biotin = ATP = SAM N-acetylglucosamine-1-P are obligate intracellular microbes with fragmented genomes and Dihydroxy GpsA sn-Glycerol D-ribose 5-P D-Ribulose 5-P CMP-Kdo previously characterized) required to compensate for the patchwork Rickettsia -acetone = acetyl-CoA = [4Fe-4S] predicted to be imported Rickettsia Imported Metabolite Exact Mass of Metabolite (KDa) 3-phosphate RpiB GlmU CoaE PccA BCCP BirA phosphate = NADPH = CTP diminished metabolic capabilities. They are found widely distributed metabolic network (Fig. 2). Fatty acid and glycerphospholipid synthesis initiate from 0 1 2 3 4 5 6 7 8 = NADH = G3P A V = UTP = L-Serine Flavin adenine dinucleotide ? UDP-glucose UDP-N-acetyl-alpha-D-glucosamine among vertebrate hosts and include several serious human pathogens. host precursors (Fig. 3), and the import of both isoprenes and terpenoids is required for (CoA) Biotin- Dephosphocoenzyme A PccABCCP (Host derived?) Ugd FnlA WecB WecA LpxA MurA Acyl carrier Lysophosphatidic Nicotinamide adenine dinucleotide AcpS protein (AcpP) acid (LPA) Phosphatidylcholine UDP-glucuronate UDP-alpha-D UDP-N-acetyl-D N-acetyl-alpha-D-glucos- UDP-3-O-(3- UDP-NAG- Although the metabolic intractability of the rickettsiae has been studied the synthesis of ubiquinone and the lipid carrier of Lipid I and O-antigen (data not SAH - PccA PC HCO 3 -galactose -mannosamine aminyl-diphospho-ditrans, hydroxytetradecanoyl) enolpyruvate 5,6,7,8-Tetrahydrofolate Holo-ACP PlsC choline octacis-undecaprenol -N-acetylglucosamine for decades, few details are known about how rickettsiae acquire the shown). Unlike bacterial symbionts of arthropods, rickettsiae lack the capability for de EamA A Arthropods Thiamine diphosphate PccB Carboxy- Pld Malonyl-CoA biotin- Pld V Vertebrates S-Adenosyl-L-methionine FabD Phosphatidic acid Lipid I metabolites required for their survival. In this study, we integrate recent novo synthesis of B or most other cofactors (Fig. 4); consequently, we stress PccABCCP Essential metabolite Kdo 2 -lipid A extension; O-antigen polymerization O-antigen carrier Lipid A (Peptidoglycan) Pld de novo synthesis Tlc5 Hexadecanoyl-ACP Pyridoxine phosphate Malonyl-ACP CdsA genomic data with historical analyses to construct a comprehensive, the use of “endoparasite” over “endosymbiont” for non-pathogenic rickettsiae. Cell Synthesized from BioY (cycle 7 product) ethanolamine essential metabolite Biotin ADP FabH (cycles 2-8) Phosphatidyl- genus-level metabolic and transport network for the rickettsiae. envelope glycoconjugates are synthesized from three imported host sugars (Fig. 5A), Both 4-hydroxybenzoate CDP-diacylglycerol ethanolamine Figure 5B. Rickettsiae possess a complete TCA cycle, but require a Variable Guanosine 5'-triphosphate (Host derived?) Tlc1 β-Ketoacyl-ACP Acyl-ACP with a range of additional host-acquired metabolites fueling the TCA cycle (Fig. 5B). Six Adenosine 5'-triphosphate FabF range of host-acquired cofactors to fuel it (ATP, CoA, Glx, etc). Uridine 5'-triphosphate PgsA PssA FabG Composition Cytidine 5'-triphosphate FabI Peptidoglycan, biosynthetic pathways contain pathway holes (Fig. 6A; see METHODS), and similar Octanoyl-ACP Phosphoenol- FabZ PE = 60-70% CMP-Kdo (LPS) pyruvate (PEP) Guanosine 5'-diphosphate Tlc4 β-hydroxyacyl-ACP β-enoyl-ACP (cycle 3 product) Phosphatidyl- Phosphatidyl- N-Succinyl-LL-2,6- N-Succinyl-2-L-amino PG = ~20% diaminopimelate -6-oxoheptanedioate patterns in taxonomically diverse bacteria (Fig. 6B) suggest atypical chemistry or Guanosine 5'-monophosphate glycerophosphate L-serine ? Adenosine 5'-monophosphate LipB PS PC = ~15% PpdK DapE Glycero- PdhA CONCLUSIONS utilization of novel metabolites. A paucity of characterized and predicted transporters Uridine 5'-monophosphate PS = trace Suc-CoA DapD phospholipids PdhB PgpA Psd Pyruvate Known to be (2Z,6Z)-Farnesyl diphosphate Protein N6- CL = trace Tlc2, Tlc3 LL-2,6-Diaminopimelate 2,3,4,5-Tetra- Collectively, our work provides a detailed metabolic profile of rickettsial (see Fig. 2) emphasizes the gap in our knowledge concerning how rickettsiae imported Isopentenyl diphosphate (3R)-3-Hydroxy- (3R)-3-Hydroxy- (octanoyl)lysine 2-Hydroxy- tetradecanoyl-ACP hexadecanoyl-ACP Phosphatidyl- Phosphatidyl- hydrodipicolinate TdcB ethyl-ThPP Dimethylallyl diphosphate glycerol ethanolamine ThDP (cycle 6 product) (cycle 7 product) LipA DapF parasites and highlights aspects of reductive genome evolution and import host metabolites, many of which are large and not known to be transported by COG2984 PHB UDP-glucose PG PE L-Serine COG4120 DapB or PdhA Lipo- N-Acetyl-a-D-glucosamine 1-phosphate transacylation addiction to host cell metabolites. We show that NAG-1-P meso-2,6-Diaminopimelate PdhB amide-E the core biosynthesis any described bacteria. COG1101 Protein N6- ? reactions; PhbC D-Ribose 5-phosphate Pi HTPA (lipoyl)lysine Aas phospholipase Phosphorylcholine GlmU_N A1 degradation GlyA hubs in Rickettsia have been supplanted by elaborate thievery of / sn-Glycerol 3-phosphate GlpT Cardiolipin 2-Acyl-sn- DapA (R)-3-hydroxy- / Peptidoglycan butyryl-CoA S-Acetyl- UDP-NAG CL glycero-3- dihydro- Dihydroxyacetone phosphate + Lpd host metabolites to complement a patchwork metabolic network. We ACKNOWLEDGEMENTS H E2 subunits of phospho- L-Aspartate 4-semialdehyde lipoamide-E (S)-Malate SLC5sbd 2-oxoglutarate (Host derived?) ethanolamine L-Glycine LpxA and pyruvate OXPHOS PhbB / Pyruvate YfdV-1, Dihydrolipo- also reveal a startling degree of functional conservation across all 84 Supported with funds from National Institute of Health/National Institute of Allergy and + dehydrogenase YfdV-2 Na ? known to be imported predicted to be imported Asd Fatty Acids amide-E Biosynthetic pathways Acetate Acetoacetyl-CoA

UDP-3-O-(3-hydroxy / tetradecanoyl)-N- PdhC sequenced Rickettsia genomes regardless of host or pathogenicity. Infectious Diseases grants (R01AI017828, R01AI126853, R21AI26108, T32AI095190, L-Tryptophan 4-Phospho-L-aspartate MaeB Cofactors/cosubstrates acetylglucosamine Hexadecanoyl-ACP A L-Tyrosine (cycle 7 product) PhbA Ribonucleotides Cationic aa transporters LPS and T32AI007540. We are grateful to Dr. Lucy Weinert (University of Cambridge) for L-Arginine + + Acetyl-CoA Finally, we identify areas of future research aimed at understanding the H H LpxC Octadecanoyl-ACP CtaA LysC Terpenoid backbone L-Phenylalanine UDP- (cycle 8 product) 2-O-(2-hydroxy / sharing the unpublished genomes of Rickettsia species isolated from the ladybird beetle Cell envelope glycoconjugates Heme O AcnA Citrate Oxaloacetate host/parasite interface and elucidating the chemical dependency that L-Histidine AdiC PotE hexadecanoic), RpiB, KdsD, KdsA, KdsB or UDP-3-O-(3-hydroxy GltA Mdh Glycerophospholipids L-Methionine tetradecanoyl)-D- 3-O-(3-hydroxy Kdo2 Lauroyl-Kdo2 Kdo 2 - SucA glucosamine tetradecanoic)- -lipid IV(A) -lipid IV(A) lipid A defines the Rickettsia obligate intracellular lifestyle. Adalia bipunctata and the parasitic ciliate Ichthyophthirius multifiliis. Pyr/Ac-CoA-TCA cycle L-Glutamate LpxD D-glucosamine LpxI, LpxB, LpxK, WaaA LpxL LpxJ CtaB 3-Carboxy- Isocitrate (S)-Malate Proteogenic amino acids L-Lysine Glutamate 1-hydroxy AspC L-Glutamine propyl-ThPP GlnHPQ Aspartate GlnA Transporters L-Aspartate FumC

HemH ThDP Icd / ABC MFS L-Asparagine GltP Lipo- Glutamine L-Leucine Protoporphyrin SucA amide-E GdhX AAA VUT + 2-Oxoglutarate Fumarate Figure 1. Rickettsia metabolic network reconstruction highlights reductive genome evolution and an addiction to host cell metabolites. L-Isoleucine H Figure 4. Rickettsiae lack the capacity for de novo folate synthesis, / / AEC DMT L-Cysteine COG2984 HemJ SdhD S-Succinyl- / / / L-Threonine COG4120 but may use FolE to initiate queuosine synthesis from imported GTP. Succinyl-CoA Succinate SdhC APC SSS Protoporphyrinogen IX dihydro- Lpd FAD, FMN L-Valine SucC SucD SdhA SdhB FAD FMN COG1101 lipoamide-E PLP DAACS L-Proline 2,5-diamino-6-(5'-triphosphoryl FolE 2,5-diaminopyrimidine FolE Formamidopyrimidine FolE HemF PLP Na+ ? -3',4'-trihydroxy-2'-oxopentyl)- GTP Dihydrolipo- = ATP = CoA = PEP RNA DNA Pyrimidines RNA DNA L-Serine nucleoside triphosphate nucleoside triphosphate HemN ALAS + Pyrimidines ProP1-7 amino-4-oxopyrimidine amide-E = ADP = SAM = Fe2 Predicted transporter L-Alanine DNA DNA SLC5sbd + HK SucB = UTP = PLP = [4Fe-4S] Coproporphyrinogen III 5-Aminolevulinate + Characterized transporter Glycine H + (fusion protein) QueD QueE QueC = NAD = Lipoate GLY FolE 6-carboxy-5,6,7,8- 7-carboxy-7- 7-cyano-7- = Ubiquinone GTP tetra-hydropterin carbaguanine carbaguanine = NADH = 5,6,7,8-Tetrahydrofolate HemB + D-ribose 5-P HemE = NADP = 5,10-Methylene-THF RNA UDP-glucose RNA LEFT: Metabolites are grouped according to biosynthetic pathways (see inset at BOTTOM UMP UTP GDP 7,8-dihydroneopterin QueF HemD HemC = NADPH = trans,trans-Farnesyl-PP Lipopolysaccharide Lipopolysaccharide Queuosine biosynthesis Uroporphyrinogen III Hydroxymethylbilane Porphobilinogen = FAD = (3R)-3-Hydroxy-tetradecanoyl-ACP RNA RNA LEFT), with red ellipses depicting metabolites previously shown to be imported. The 3'-triphosphate CTP GMP = FADH 2 = di-trans,octa-cis-undecaprenyl-P NAG-1-P 34 DNA DNA remaining 30 metabolites are predicted to be imported in the current study. Phosphatase queuosine 7-aminomethyl- Peptidoglycan Peptidoglycan L-Ala Purines (G) ? (in tRNA) 7-carbaguanine Purines CENTER: Biosynthesis capabilities for each metabolite in arthropod and vertebrate 7,8-dihydroneopterin PTPS III epoxy- Folate biosynthesis ? Tgt queuosine genomes (further described in inset at TOP LEFT). ThDP Purines (A) FolB Pentose Phosphate ThDP (in tRNA) DHAP unknown unknown pterin epoxy- 7-aminomethyl-7- Terpenoid backbones Queuosine Terpenoid backbones substrate 34 34 Figure 6A. Rickettsiae contain six pathway holes in otherwise AMP RIGHT: Within each group, metabolites are ranked by exact mass. Dashed lines similar to HMDHP; queuosine deaza-guanosine plus the probable (in tRNA) QueA (in tRNA) Glycerophospholipids Glycerophospholipids connect metabolites with their known or predicted transport systems (see inset at 6-hydroxymethyl-7, “frC” elimination of tri- conserved metabolic pathways, suggesting alternative chemistries. FPP IPP 8-dihydropteridin phosphate (PTPS- BOTTOM LEFT). Phylogenomics analysis indicates these transporters are highly (HMDHP) like; EC 4.2.3.12) = [4Fe-4S] = FADH2 Asp 2 3 4 5 6 7 8 meso-DAP DMAPP ATP 4-aminobenzoate = SAM = FAD m-DAP ABC, ATP-Binding Cassette; AAA, ATP:ADP Antiporter; DMT, + MEP/DOXP conserved in rickettsial genomes. FolK/P (pABA) FolK/P = ATP = NAD - No (DapC, ArgD) to convert N-Succinyl-L-2-amino-6-oxopimelate (6) to N-Succinyl-LL-2,6-diaminopimelate (7) RNA DNA RNA DNA Drug/Metabolite Transporter; VUT, Uptake Transporter; AEC, Auxin Efflux Carrier; MFS, Major = ADP = NADH GTP D-Ribulose 5-phosphate 2 3 4 CMP-Kdo THF G3P 7,8-dihydropteroate 7,8-dihydropteroate = dTMP = L-Glutamate CMP-Kdo Facilitator Superfamily; APC, Amino Acid-Polyamine-Organocation; DAACS, Dicarboxylate/Amino = dUMP = L-Methionyl-tRNA - No enzyme (KdsC) to convert 3-Deoxy-D-manno-octulosonate 8-P (3) to 3-Deoxy-D-manno-octulosonate (4) PHBA THF Acid:Catio (Na+ or H+) Symporter; SSS, Solute:Sodium Symporter. = NADP+ = N-Formylmethionyl-tRNA FolC Dihydroxyacetone phosphate 2 3 4 CDP-diacylglycerol PDC PDC Pyruvate = NADPH = L-Serine CDP-DG = PLP = Glycine - No enzyme (PlsB, PlsX/Y) to convert sn-Glycerol 3-phosphate (2) to Lysophosphatidic acid (3) + Lipoate Lipoate NAD 7,8-dihydrofolate PHB PHB TdcB ThDP Isopentenyl diphosphate (IPP) + Dimethylallyl diphosphate (DMAPP) Geranyl diphosphate (GPP) GSH + METHODS FolA / One-carbon pool by folate known to be imported TERP-BB IPP + GPP trans,trans-Farnesyl diphosphate (FPP) S G predicted to be imported - No enzyme (IspA, IdsA, GGPS) to convert IPP and DMAPP into GPP, or IPP and GPP into FPP NAD 8 8 Diaminopimelate Diaminopimelate GSH FolD Pyruvate Orthologous gene families (n=3,576) were constructed from 84 5,6,7,8-tetrahydrofolate 5,10-methylene- (THF) Chrorismate 2 3 [-] 4 5 6 7 8 9 Ubiquinone-8 (CoQ ) GlyA tetrahydrofolate / UBI-8 8 sequenced rickettsial genomes using fastortho, a modified version of (storage?) - No to convert Chorismate to 4-Hydroxybenzoate (2), or 2-Octaprenylphenol (4) to 2-Octaprenyl-6-hydroxyphenol (5) Ubiquinone (CoQ ) Ubiquinone (CoQ ) Fatty Acid Fatty Acid / Q FolC (import?) Gln Gln E GTP 2 3 4 5 6 7 queuosine 34 (in tRNA) TCA cycle TCA cycle OrthoMCL, and a subset of 149 completely conserved single-copy FolD YgfA 5-formyltetra- QUEU 10-formyl- 5,10-methenyl- - No enzyme (QueG) to convert epoxyqueuosine 34 (in tRNA) (7) to queuosine 34 (in tRNA) Glu Malonyl-CoA Glu Malonyl-CoA Tetrahydropteroyldiglutamate THF tetrahydrofolate tetrahydrofolate hydrofolate D Fmt ThDP families was used to estimate a genus-level phylogeny. All protein (Folinic acid) OXPHOS OXPHOS Malate / Asp Asp FolC GlyA? / sequences (n=109,191) were submitted to BlastKOALA and KEGG / FolD Porphyrins Porphyrins Tetrahydropteroyl-[ -Glu] 5,10-methylene- Figure 6B. Several rickettsial pathway holes ( ) are also common n tetrahydrofolate Biotin ThyX / Coenzyme A dephospho-CoA Biotin classifications were subsequently extracted (kcompile), transferred across a taxonomically diverse set of bacteria. across orthologous families (ktransfer), and used for metabolic network Queuosine biosynthesis Folate biosynthesis One carbon pool by Folate CMP-Kdo Alphaproteobacteria n = 442 reconstruction (kreconstruct). Rickettsial pathway holes (missing gene in TERP-BB Gammaproteobacteria LEFT RIGHT n = 37 : Theoretical Rickettsia metabolic network in the absence of imported host : Reconstructed Rickettsia metabolic network including imported metabolites. 80 1 Betaproteobacteria metabolites. Rickettsia metabolic pathways have been “supplemented” with typical Pathways removed from LEFT have been purged from Rickettsia genomes throughout an otherwise complete metabolic pathway) were confirmed by blastp, and 70 Deltaproteobacteria Gram-negative biosynthetic pathways to create a complete metabolic network. evolution, a consequence of metabolite pilfering from the eukaryotic host. their taxonomic distribution assessed by converting into feature vectors 60 QUEU 4 50 n = 61 Epsilonbacteria 7 and pattern matching against all complete bacterial genomes in KEGG 40 30 1 16 Chlamydiales 30 (khole). Transporter systems were assembled in an iterative process UBI-8 1 The network focuses on the biosynthetic pathways discussed in the text. For brevity, the pathways for most amino acids Abbreviations: FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; 20 n = 74 1 Spirochaetes have been omitted. Red stars indicate six pathway holes found in this study (see Fig. 6). Red ellipses indicate 82 m-DAP PLP, ; GLY, glycolysis; ThDP, thiamine diphosphate; involving bioinformatic predictions (TransportDB and TCDB), manual 10 2 n = 315 Bacteroidetes

metabolites known to be imported by Rickettsia species. Yellow ellipses indicate metabolites predicted to be imported MEP/DOXP, non-mevalonate terpenoid biosynthesis; THF, No. Rickettsia genomes (out of 84 total genomes) 0 evaluation, and comparative genomics analyses. Custom software Actinobacteria based on this study. Import of S-Adenosyl-L-methionine, phosphorylcholine, and the majority of amino acids are not 5,6,7,8-tetrahydrofolate; GSH, glutathione; NAD, nicotinamide adenine included in the reconstruction. Pathway lines are highlighted red to indicate cofactors that are synthesize directly from folE queD queE queC queF tgt queA folB “frC” folKP folC folA thyX fmt glyA tdcB folD ygfA Firmicutes dinucleotide; PHB, polyhydroxybutyrate; PDC, pyruvate dehydrogenase complex. packages developed for this study are freely available from the keggerator Homolog found in imported metabolites. Yellow pathway lines with red highlighting indicate cofactors that are directly imported from the host. CDP-DG repository on github. Conserved (full length) Truncated or fragmented n = 433 Other familial ancestor

This work is dedicated to the late Dr. Herbert H. Winkler (1939-2016, former Professor of Microbiology and Immunology at the University of South Alabama College of Medicine), whose pioneering work on Rickettsia metabolism paved the way forward, and in memory of Chris Cornell (1964-2017), author of “Black Hole Sun”.